臺灣博碩士論文加值系統

哺乳類細胞內的氧化磷酸化系統是由五個酵素蛋白複合體所構成。五個酵素蛋白複合體中，粒線體酵素複合體I是最大且最複雜的,它含有許多未定義的次單元,其完整的結構也尚未解開。哺乳類的粒線體酵素複合體I包含45個次單元,其中7個次單元由粒線體基因體組所表達。剩餘的次單元則均由細胞核基因體組所表現，並被運送入粒線體執行他們的功能。 NADH dehydrogenase (ubiquinone) Fe-S protein 8，簡稱NDUFS8，是一個細胞核基因體組所表現的粒線體酵素複合體I的核心蛋白，其含有兩個[4Fe-4S]鐵硫中心，在電子傳遞鏈中扮演重要角色。 NDUFS8突變已知會造成Leigh症候群，並且引起粒線體酵素複合體I的缺失。在這個研究中，主要是利用核糖核酸干擾(RNAi)技術抑制T-REx293細胞中NDUFS8基因的表現，藉此來探討NDUFS8的功能。實驗結果顯示抑制NDUFS8會使細胞生長遲緩、耗氧能力下降、增加活性氧 (ROS) 的產生。利用高解析度透明非變性聚丙烯醯胺凝膠電泳 (HrCNE) 檢測粒線體酵素複合體I的完整性顯示抑制NDUFS8的表現不會影響粒線體酵素複合體I的組成，但減弱其氧化NADH的活性。在NDUFS8受抑制的細胞中表現NDUFS8可改善細胞耗氧能力及粒線體酵素複合體I氧化NADH的活性。另外，各種NDUFS8刪除及與綠螢光蛋白(EGFP)融合的載體被建構來探尋NDUFS8的粒線體標的訊號。實驗結果顯示NDUFS8的胺端18個胺基酸長的片段即擁有將綠螢光蛋白(EGFP)送入粒線體的能力，這比MitoprotII軟體所預測的34個胺基酸還要短。有趣的是，胺端被刪除的NDUFS8蛋白意外的進入細胞核內的特定區域，推測可能有細胞核標的訊號潛藏在NDUFS8序列中。這個研究顯示NDUFS8在粒線體酵素複合體I的活性上扮演重要的角色，而NDUFS8的粒線體標的訊號存在與否，將會決定蛋白在細胞內的位置是在粒線體或細胞核。

Oxidative phosphorylation system in mammalian cells contains five enzyme complexes. Among them, mitochondrial complex I is the biggest and the most complicated, with many undefined subunits and has no resolved complete structure. Mammalian mitochondrial complex I comprises of forty-five subunits, and seven of them are encoded by the mitochondrial genome. The remaining subunits are encoded by the nuclear genome and imported into mitochondria to perform their functions. NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is one of the nuclear-encoded mitochondrial core proteins of complex I. It contains two tetranuclear iron-sulfur clusters and plays an important role in electron transport. Mutations on NDUFS8 have been found to cause Leigh syndrome with mitochondrial complex I deficiency. In this study, RNA interference technique was used to knock down the NDUFS8 expression in T-REx293 cells to investigate the function of NDUFS8. Experimental results demonstrated that reducing expression of NDUFS8 would retard the cellular growth rate, slow down the oxygen consumption efficiency and increase the production of reactive oxygen species (ROS). Using high resolution clear native gel electrophoresis (HrCNE) for investigating the integrity of mitochondrial complex I revealed that knockdown of NDUFS8 would not affect the assembly of mitochondrial complex I but reduce its NADH oxidation activities. Restoration of NDUFS8 in a suppressed cell line improved the ability of oxygen consumption and NADH oxidation of complex I. In addition, various deletion and fusion constructs of NDUFS8 were generated to characterize the mitochondrial targeting sequence of this protein. The results revealed that the N-terminal fragment of 18 residues possessed the ability to import EGFP into mitochondria, which is shorter than the prediction of 34 amino acid residues proposed by MitoprotII program. Interestingly, there was also an unexpected result that all of the N-terminal deletion constructs of NDUFS8 protein were located in a specific region of nuclei. It was speculated that there is a nuclear localization signal hiding in NDUFS8 sequence. This study demonstrated that NDUFS8 play an essential role in complex I activity, and the mitochondrial targeting sequence of NDUFS8 existing or not will determine the subcellular localization in mitochondria or in nuclei.

中文摘要 i
Abstract ii
Abbreviations vii
Introduction 1
Materials and methods 16
Results 24
A. The functional study of NDUFS8 24
1. Reduced and reconstituted expression of NDUFS8 in T-REx293 cells 24
2. Growth capability was diminished in NDUFS8 suppression cells and NDUFS8 rescue cells 25
3. Oxygen consumption rate of respiratory chain declined in NDUFS8 suppression cells and recovered in NDUFS8 rescue cells 26
4. Reactive oxidative species generation was promoted in NDUFS8 suppression cells and NDUFS8 rescue cells 26
5. NADH oxidation activity of mitochondrial complex I was affected by NDUFS8 kncodown but complex I assembly was not 27
B. Characterization of mitochondrial targeting sequence of NDUFS8 29
1. Mapping mitochondrial targeting sequence of NDUFS8 by fused with EGFP 29
2. Mapping mitochondrial targeting sequence by N-terminal truncated NDUFS8 30
3. Mapping the nuclear localization signal in the N-terminal truncated NDUFS8 fragment 31
Discussion 33
A. The functional study of NDUFS8 33
B. Characterization of mitochondrial targeting sequence of NDUFS8 36
Tables 40
Figures 43
Figure 1. Mutiple sequence alignment of the complex I NDUFS8 subunit. 43
Figure 2. The knockdown efficiency of NDUFS8 RNAi. 45
Figure 3. Growth curves of T-REx293, shRNA-A4, shRNA-B3 shRNA-C3 and shRNA-C3-Rescue cells in glucose-containing DMEM (A) and in galactose-containing DMEM (B). 46
Figure 4. Oxygen consumption measurement in T-REx293, shRNA-A4, shRNA-B3, shRNA-C3 and shRNA-C3-Rescue cells by Mitocell S200 micro respirametry system. 47
Figure 5. ROS generation assay detecting H2O2 level in T-REx293, shRNA-A4, shRNA-B3, shRNA-C3 and shRNA-C3-Rescue cells by FACScan flow cytometer. 48
Figure 6. Mitochondrial complex I assembly and in-gel catalytic assay in T-REx293, shRNA-A4, shRNA-B3 shRNA-C3 and shRNA-C3-Rescue cells by HrCNE. 50
Figure 7. Characterization of the mitochondrial targeting sequence of NDUFS8 by various N-terminal fragments fused with EGFP. 53
Figure 8. Characterization of mitochondrial targeting sequence of NDUFS8 by different length of N-terminal deletion constructs tagged with c-myc. 55
Figure 9. Mapping the nuclear localization signal hiding in NDUFS8 sequence by different segment of NDUFS8 combined with LacZ. 56
References 57
Appendixes 63

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